Our long-term objective is to determine the contribution of acid-active hyaluronidases (hyases) to sperm function and reproduction in general. The purpose of this R03 application is to use available Hyal2 and Hyal3 null mice to investigate the role of two novel sperm hyases, HYAL2 and HYAL3 in mouse sperm function, and subsequently determine their presence in human sperm. Of the 6 mammalian hyases that catalyze the breakdown of hyaluronic acid (HA) in the extracellular matrix, 5 are known to be abundantly expressed in the testis. Of these 5, 2 acid-active and 3 neutral-active, only the latter which are multifunctional proteins exemplified by Sperm Adhesion Molecule 1 (SPAM1 or PH-20) are considered to be sperm hyases. There have been no reports of the presence of the 2 acid-active hyases in sperm, and our preliminary studies suggest that in humans and mice they (HYAL2 and HYAL3) are present in sperm and may contribute to the overall hyase activity that is essential in fertilization. Moreover, they may be responsible for sperm-zona binding, which has been attributed to SPAM1, but recently shown not to be so in mice. Importantly, HYAL2 may perform an essential unique role in that its deficiency which is not compensated for by family members (unlike other hyases) leads to infertility. Based on our preliminary data, our central hypothesis is that HYAL2 and HYAL3 are present in mouse and human sperm where they play both enzymatic and non- enzymatic roles and that HYAL2 has a unique function (not compensated for by other hyases) in fertilization. Due to our experience working with hyases, our supportive preliminary data, and our available mutants we are ideally positioned to pursue and accomplish the following two Specific aims to test our central hypothesis: I. Determine if HYAL2 and HYAL3 are present in mouse sperm and epididymis where they can be acquired by sperm, and the basis of the infertility of Hyal2 nulls. A) Localize and determine the subcellular distribution of the proteins in wild-type (WT) sperm and compare hyase activity at pH 3-7 with those of the nulls. B) Assess the andrological parameters and the fertility index of Hyal2 nulls by natural mating to WT. C) Compare the ability of the nulls and WT sperm to bind HA, penetrate the cumulus, and perform the acrosome reaction. D) Compare the zona-binding affinities of HYAL2 and HYAL3 with other hyases. E) Investigate the presence of Hyal2 and Hyal3 mRNAs and proteins in the epididymis/epididymal fluid and if the proteins can be acquired on sperm plasma and inner acrosomal membranes after in vitro exposure. II. Determine if HYAL2 and HYAL3 are present and are catalytically active in human sperm. A) Assess the subcellular distribution of the proteins in human sperm. B) Analyze human sperm hyase activity at pH 7, 3 and 4 which is the optimum for acidic hyases. Clearly a study of these hyases in sperm is highly warranted since the characterization of enzyme-specific functions is crucial for the diagnosis and treatment of sperm pathology in the clinic. PUBLIC HEALTH RELEVANCE: Due to the essential roles played by hyaluronidases in sperm-egg interactions, there are important implications of this work for human fertility. It will identify and help establish a role for two novel sperm hyases in humans where SPAM1 has so far been the only one reported. Critical levels of hyases are required for the success of in vitro fertilization (IVF) where recombinant SPAM1 has been used. HYAL2 and 3, acidic hyases, are also expected to play a critical role in IVF and the recombinant proteins can now be used to advance assisted reproductive technology via their addition to the sperm surface, to treat sperm pathology.